Expression Profile of Genes from 12P in Testicular Germ Cell Tumors Of
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Oncogene (2003) 22, 1880–1891 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2–p12.1 S Rodriguez1,7, O Jafer1,7, H Goker1,6, BM Summersgill1, G Zafarana2, AJM Gillis2, RJHLM van Gurp2, JW Oosterhuis2, Y-J Lu1,3, R Huddart4, CS Cooper5, J Clark5, LHJ Looijenga2 and JM Shipley*,1 1Molecular Cytogenetics, Institute of Cancer Research, Sutton, Surrey, UK; 2Pathology/Laboratory for Experimental Patho-Oncology, Erasmus MC University Medical Center Rotterdam, Daniel den Hoed Cancer Center, Josephine Nefkens Institute 3000 DR Rotterdam, The Netherlands; 3Section of Paediatric Oncology, Institute of Cancer Research, Sutton, Surrey, UK; 4Academic Department of Urology, The Royal Marsden National Health Service Trust and Institute of Cancer Research, Sutton, Surrey, UK; 5Cell Transformation, Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton Surrey, UK Gain of 12p material is invariably associated with Keywords: testicular germ cell tumors of adolescents testicular germ cell tumors (TGCTs) of adolescents and and adults; isochromosome 12p; amplification; micro- adults, most usually as an isochromosome 12p. We array expression profiling; comparative expressed analyzed TGCTs with i(12p) using a global approach to sequence hybridization; candidate genes expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2–p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype Introduction showed higher levels of expression than seminomas. Notably, 12p11.2–p12.1 is amplified in about 10% of Testicular germ cell tumors (TCGTs) of adolescents and TGCTs and CESH analysis of such amplicon cases adults comprise 1% of all malignancies in men and have showed high levels of overexpression from this region. been reported as increasing in incidence over the last Microarray analysis, including cDNA clones representing several decades in Caucasian populations (Bosl and most UniGene clusters from 12p11.2–p12.1, was applied Motzer, 1997). Although the majority of TGCTs are to DNA and RNA from 5 TGCTs with amplification of curable, these tumors are the leading cause of cancer 12p11.2–p12.1 and seven TGCTs with gain of the entire death in young men (Bosl and Motzer, 1997). The short arm of chromosome 12. Expression profiles were tumors are classified into two main subtypes; the consistent with the CESH data and overexpression of seminomas and the nonseminomas. Various character- EST595078, MRPS35 and LDHB at 12p11.2–p12.1 was istics including their sensitivity to cis-platinum-based detected in most TGCTs. High-level overexpression of treatment and genetic changes suggest a common BCAT1 was specific to nonseminomas and overexpression etiology for these subtypes. Seminomas frequently of genes such as CMAS, EKI1, KRAS2, SURB7 and present clinically at a more advanced age than various ESTs correlated with their amplification. Genes nonseminomas, suggesting a slower rate of progression such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were (Oosterhuis et al., 1989). TGCTs are generally consid- also overexpressed. The overexpressed sequences identi- ered to be derived from carcinoma in situ (CIS), which fied, particularly those in the region amplified, represent develop in utero and do not progress until after puberty candidate genes for involvement in TGCT development. (reviewed by Rorth et al., 2000). ONCOGENOMICS Oncogene (2003) 22, 1880–1891. doi:10.1038/sj.onc.1206302 A striking feature of all invasive TGCTs is gain of material from the short arm of chromosome 12. This is because of an isochromosome 12p in 80% of cases (reviewed by Sandberg et al., 1996). In approximately *Correspondence: JM Shipley, Molecular Cytogenetics, Male Urolo- 10% of primary TGCTs, amplification in the 12p11.2– gical Cancer Research Centre, Institute of Cancer Research, 15 p12.1 region has been reported (Suijkerbuijk et al., 1993; Cotswold Road, Sutton, Surrey SM2 5NG, UK; Mostert et al., 1996; Korn et al., 1996; Rao et al., 1998; E-mail: [email protected] Summersgill et al., 1998a; Roelofs et al., 2000). This has 6Current address: Section of Gene Function and Regulation, Institute been predominantly found in seminomas although it has of Cancer Research, Chester Beatty Laboratories, Fulham Road, London, UK been seen in nonseminomas but in a more heterogeneous 7These authors contributed equally to this work cellular pattern. The amplification is present mainly in Received 24 October 2002; revised 6 December 2002; accepted 6 seminomas without an i(12p) although gain of the entire December 2002 short arm of 12p has been characterized in addition to 12p Expression profiling in testicular germ cell tumors S Rodriguez et al 1881 the amplification (Roelofs et al., 2000). Seminomas with The CESH profiles of cases with amplicons (aSE1, amplification at 12p11.2–p12.1 have been associated aSE2) showed a similar pattern although the maximum with an earlier age of clinical presentation than ratios of fluorescence intensity were higher than the seminomas with gain of the entire 12p, and have been nonamplicon cases. Direct comparison of seminomas related to reduced sensitivity of the tumor cells to with and without 12p11.2–p12.1 amplification (aSE4 vs undergo apoptosis (Roelofs et al., 2000). Neither gain SE6 and aSE4 vs SE7) showed relative overexpression nor amplification of 12p material has been detected in from 12p peaking at the region of amplification (Figure CIS, although many of the other chromosome imbal- 2d, e). Comparing two nonseminoma without evidence ances associated with TGCTs were already present in for amplification of the 12p11.2–p12.1 region (NS9 vs CIS adjacent to invasive tumor (Rosenberg et al., 2000; NS10) showed no differential expression on 12p (data Summersgill et al., 2001a). This indicates that 12p gain is not shown). These data show that relative overexpres- associated with the progression of CIS to invasive sion of genes from 12p11.2 to 12p12.1 is likely to be disease. important in all TGCT, irrespective of amplification. It is likely that the amplification leads to over- expression of genes that are involved in TGCT progres- sion, as has been found in other tumor types (Hayes and Thor, 2002). However, as the gain of the entire short arm of chromosome 12 is such a consistent feature and Identification of amplified clones by CGH microarray is found in addition to amplification of the 12p11.2– analysis p12.1 region, it is likely that genes outside the amplified region on 12p may also be involved. To define the region of amplification at the level of To further understand the role of 12p in the cDNA clones, we applied microarray CGH analysis to development of TGCTs, we investigated the overall cDNA clones for chromosome 12 (www.icr.ac.uk/ expression patterns along chromosome 12 in TGCTs rodriguez/). The microarray data for the cell line with and without amplification at 12p11.2–p12.1. We GCT27 and cases known to have uniform gain of 12p have profiled the expression patterns along this chromo- material were analyzed, and examples of the profiles some using two approaches: (i) using a novel approach, obtained are shown in Figure 3a–d. we developed, which targets chromosome preparations The log 2 ratio of fluorescence intensities for amplified and indicates chromosome regions containing genes that clones was generally in excess of 1.5, whereas 1.5–0.3 are relatively under- and overexpressed (Lu et al., 2001); was considered as gain and 070.3 as normal. The and (ii) using microarray analysis to identify over- 1og 2 ratio of fluorescent intensities for the clones on represented and overexpressed genes and expressed 12p outside the region amplified in the amplicon sequences in TGCTs with gain of the entire short arm cases was generally greater than 0.3 and greater than of chromosome 12 and TGCTs that also have amplifica- those for clones on 12q (070.3). This suggests tion at 12p11.2–p12.1 (Figure 1). gain of 12p material in addition to the amplification and is consistent with the copy number of YAC markers determined by interphase FISH analysis in this study and previously (Zafarana et al., 2002). Results The results of the CGH microarray analysis of the five Comparative expressed sequence hybridization (CESH) TGCTs with 12p11.2–p12.1 amplicons are presented in analysis Figure 3e–h, and the clones amplified in each case are indicated in Tables 2 and 3. The profiles for aSE2 and CESH analysis was carried out to profile the expression aSE4 suggest two maximum peaks of amplification changes along the length of chromosome 12 in various interspersed with a lower level of gain at around TGCTs with gain of the entire 12p region and also 24MB (Figure 3h, j). This is consistent with the pattern amplification at 12p11.2–p12.1 (Table 1). Direct com- of amplification determined by microarray data with parison of the human foreskin fibroblast cultures and/or BAC clones for the same cases (Zafarana, unpublished normal testicular parenchyma used as controls showed data). little difference along the length of 12p, except for a A few clones were identified as amplified although small dip in the very telomeric region suggesting relative according to the databases mapped outside the 12p underexpression from genes in this region in normal amplicon region on 12p or on other chromosomes. testicular tissue (Figure 2a). CESH analysis of the cell These clones correspond to two ESTs (ID number lines in Table 1, which are all of nonseminomatous 230420, 122163) and to a gene from our geneset named origin, showed relative overexpression from the glycogen synthase (245920). We confirmed that these 12p11.2–p12.1 region, which exceeded that from other clones are likely to map to 12p and within the regions on 12p.